Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation

Bross, Peter; Andresen, Brage S.; Winter, Vibeke; Kräutle, Franz-Georg; Jensen, Thomas G.; Nandy, Andreas; Kølvraa, Steen; Ghisla, Sandro; Bolund, Lars; Gregersen, Niels

doi:10.1016/0925-4439(93)90068-c

Cited by 64 publications

(49 citation statements)

References 23 publications

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“…Among these heat shock proteins, the functional roles for GroES/GroEL, DnaK, DnaJ, and GrpE in facilitating protein folding (15,17), assembly (2,8,38), and export (14,23,34) and in minimizing protein aggregation (4,9,12,27) are well established. As shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…If this is the case, a repressor is expected to regulate the heat-induced expression of groE and dnaK operons. In support of this idea, a CIRCE-specific binding activity (5) is detected in both the crude extract and the partially purified fractions from Streptomyces coelicolor A3 (2). To determine whether a repressor is involved in controlling groE expression, we report the development of a B. subtilis system to isolate regulatory mutants that affect both groE and dnaK expression and the characterization of these mutants.…”

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confidence: 92%

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Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK

1995

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An inverted repeat sequence known as CIRCE (controlling inverted repeat of chaperone expression) in the Bacillus subtilis groE operon has been suggested to function as an operator. To identify the regulatory gene directly or indirectly involved in CIRCE-mediated heat-inducible groE expression, B. subtilis WBG2, carrying an integrated groE-bgaB transcription fusion in the amyE locus, was mutagenized. Dark blue colonies formed at 37؇C represent mutants which constitutively produce BgaB (a thermostable ␤-galactosidase) at high levels. Seven mutants (WBG101 to WBG107) were selected for further characterization. They all overproduced BgaB, GroEL, and DnaK simultaneously at 37؇C. These mutants could be restored to normal by introducing a plasmid carrying a functional copy of orf39, the first gene in the B. subtilis dnaK operon. Genomic sequencing of these mutants demonstrated that they all carried a single mutation in orf39. These mutations can be divided into three groups: (i) Gly-307 to Asp, (ii) Ser-122 to Phe, and (iii) Gly-63 to Glu. By using a binary vector system in E. coli, production of ORF39 was found to negatively regulate the expression of groE-bgaB in a CIRCEspecific manner. Under the heat shock condition, the negative regulation mediated by ORF39 was abolished. Mobility shift of the CIRCE-containing probe was also observed with the crude extract prepared from the E. coli strain that overproduced ORF39. Therefore, ORF39 is the negative regulatory factor which regulates both groE and dnaK expression in B. subtilis. It is likely to function as a CIRCE-specific repressor.Expression of Bacillus subtilis heat shock genes encoding molecular chaperones GroES, GroEL, and DnaK is regulated by a novel mechanism which differs from the well-characterized regulatory mechanism operating in Escherichia coli. In both the B. subtilis groE and dnaK operons, two sequence elements are found in the regulatory region. They are a typical A -type promoter (16,26,33) and an inverted repeat (IR) sequence designated CIRCE (controlling IR of chaperone expression) (39). CIRCE is located downstream from the transcription start site in these operons. CIRCE-like IR sequences can also be found in groE or dnaK operons from many bacteria (28). Alignment of these sequences illustrates that a consensus sequence can be generated (20,33).For groE, identical transcription start sites are observed under both heat shock and non-heat shock conditions. Mutating either the first T nucleotide in the Ϫ10 region or the third G nucleotide in the Ϫ35 region of the groE promoter results in a significant decrease in promoter activity. The activities of these mutated promoters can be restored in an allele-specific manner by specific sigA mutants at both 37 and 48ЊC (37). Transcription studies of groE with the purified A -containing RNA polymerase in vitro and a temperature-sensitive sigA mutant in vivo also support the involvement of the A -containing RNA polymerase in transcribing groE (3). Since A is the major sigma factor produced constitutively, some ot...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 92%

Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK

1995

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“…We have shown that knock-down of the HSPD1 gene by RNAi in human cells compromises folding of the mitochondrial matrix enzymes short-chain acyl-CoA dehydrogenase (SCAD) and medium-chain acyl-CoA dehydrogenase (MCAD), encoded by the ACADS and ACADM genes respectively, and mitochondrially targeted green fluorescent protein (Corydon et al 2005). In addition, disease-causing mutant variants in the medium-and short-chain acyl-CoA dehydrogenases have been shown to remain in complex with the Hsp60 chaperonin for prolonged periods of time (Saijo et al 1994;Pedersen et al 2003), and elevated chaperonin levels could partially rescue the correct folding of some of them (Andresen et al 2001;Bross et al 1993Bross et al , 1995.…”

Section: Introductionmentioning

confidence: 99%

Single-nucleotide variations in the genes encoding the mitochondrial Hsp60/Hsp10 chaperone system and their disease-causing potential

Bross

Hansen

et al. 2006

J Hum Genet

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Molecular chaperones assist protein folding, and variations in their encoding genes may be disease-causing in themselves or influence the phenotypic expression of disease-associated or susceptibility-conferring variations in many different genes. We have screened three candidate patient groups for variations in the HSPD1 and HSPE1 genes encoding the mitochondrial Hsp60/Hsp10 chaperone complex: two patients with multiple mitochondrial enzyme deficiency, 61 sudden infant death syndrome cases (MIM: #272120), and 60 patients presenting with ethylmalonic aciduria carrying non-synonymous susceptibility variations in the ACADS gene (MIM: *606885 and #201470). Besides previously reported variations we detected six novel variations: two in the bidirectional promoter region, and one synonymous and three non-synonymous variations in the HSPD1 coding region. One of the non-synonymous variations was polymorphic in patient and control samples, and the rare variations were each only found in single patients and absent in 100 control chromosomes. Functional investigation of the effects of the variations in the promoter region and the non-synonymous variations in the coding region indicated that none of them had a significant impact. Taken together, our data argue against the notion that the chaperonin genes play a major role in the investigated diseases. However, the described variations may represent genetic modifiers with subtle effects.

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“…14). We demonstrated that K304E mutant MCAD can be partially rescued from misfolding in the presence of excess GroESL and that a minor portion of the expressed K304E MCAD protein folded into the native tetrameric structure with a specific activity in the range of the wild-type enzyme (15). From these data and comparative analysis of a MCAD variant with the artificially constructed K304Q mutation, we proposed that the K304E mutation has a 2-fold effect: one on polypeptide folding, due to elimination of the positive charge in the side chain, and another one on oligomer assembly, due to the introduction of a negatively charged side chain.…”

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Effects of Two Mutations Detected in Medium Chain Acyl-CoA Dehydrogenase (MCAD)-deficient Patients on Folding, Oligomer Assembly, and Stability of MCAD Enzyme

Bross

Jespersen

Jensen

et al. 1995

Journal of Biological Chemistry

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We have used expression of human medium chain acyl-CoA dehydrogenase (MCAD) in Escherichia coli as a model system for dissecting the molecular effects of two mutations detected in patients with MCAD deficiency. We demonstrate that the R28C mutation predominantly affects polypeptide folding. The amounts of active R28C mutant enzyme produced could be modulated between undetectable to 100% ofthe wild-type control by manipulating the level of available chaperonins and the growth temperature. For the prevalent K304E mutation, however, the amounts of active mutant enzyme could be modulated only in a range from undetectable to approximately 50% of the wild-type, and the assembled mutant enzyme displayed a decreased thermal stability. Two artificially constructed mutants (K304Q and K304E1D346K) yielded clearly higher amounts of active MCAD enzyme than the K304E mutant but were also responsive to chaperonin co-overexpression and growth at low temperature. The thermal stability profile of the K304EID346K double mutant was shifted to even lower temperatures than that of the K304E mutant, whereas that of the K304Q mutant was closely similar to the wild-type. Taken together, the results show that the K304E mutation affects (i) polypeptide folding due to elimination of the positively charged lysine and (ii) oligomer assembly and stability due to replacement of lysine 304 with the negatively charged glutamic acid.Medium chain acyl-CoA dehydrogenase (MCAD\ EC 1.3.99.3) is one of four related enzymes with different chain length specificity that catalyze the initial step in mitochondrial ß-oxidation of straight chain fatty acids 0, 2). The active enzyme consists of four identical subunits, each containing one molecule ofFAD. MCAD has received particular interest, since MCAD deficiency is by far the most frequently detected inher-

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Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation

Cited by 64 publications

References 23 publications

Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK

Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK

Single-nucleotide variations in the genes encoding the mitochondrial Hsp60/Hsp10 chaperone system and their disease-causing potential

Effects of Two Mutations Detected in Medium Chain Acyl-CoA Dehydrogenase (MCAD)-deficient Patients on Folding, Oligomer Assembly, and Stability of MCAD Enzyme

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